Blood vessels are constructed by two processes: vasculogenesis, whereby a primitive vascular network is established during embryogenesis from multipotential mesenchymal progenitors; and angiogenesis, in which preexisting vessels send out capillary sprouts to produce new vessels. Endothelial cells are centrally involved in each process. They migrate, proliferate and then assemble into tubes with tight cell-cell connections to contain the blood (Hanahan, Science 277:48-50 (1997)). Angiogenesis occurs when enzymes, released by endothelial cells, and leukocytes begin to erode the basement membrane, which surrounds the endothelial cells, allowing the endothelial cells to protrude through the membrane. These endothelial cells then begin to migrate in response to angiogenic stimuli, forming off-shoots of the blood vessels, and continue to proliferate until the off-shoots merge with each other to form the new vessels.
Normally angiogenesis occurs in humans and animals in a very limited set of circumstances, such as embryonic development, wound healing, and formation of the corpus luteum, endometrium and placenta. However, aberrant angiogenesis is associated with a number of disorders, including, tumor metastasis. In fact, it is commonly believed that tumor growth is dependent upon angiogenic processes. Thus, the ability to increase or decrease angiogenesis has significant implications for clinical situations, such as wound healing (e.g., graft survival) or cancer therapy, respectively.
Antithrombin or Antithrombin III (AT3) is a single chain glycoprotein involved in the coagulation process. It is synthesized primarily in the liver with a signal peptide of 32 amino acids necessary for its intracellular transport through the endoplasmic reticulum; the peptide is then cleaved prior to secretion. Mourey et al., Biochimie 72:599-608 (1990).
AT3 is a member of the serpin family of proteins and functions as an inhibitor of thrombin and other enzymes involved in the clotting cascade. As used herein, the active native intact form of AT3 is designated the S (stressed) form (S-AT3). S-AT3 forms a tight binding complex with thrombin (markedly enhanced by the presence of heparin) and other enzymes (not all serpins have heparin affinity).
S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by a variety of enzymes, including thrombin. Evans et al., Biochemistry 31:1262912642 (1992). For example, it has been thought that thrombin binds to a reactive C-terminal loop of AT3 and the resultant complex slowly dissociates releasing thrombin and cleaving off the C-terminal loop of inactive AT3, resulting in R-AT3. R-AT3 is unable to bind thrombin and has a conformation that is quite different from that of S-AT3. The role of R-AT3 had only been known to facilitate hepatic clearance of the molecule.
Other forms of AT3, such as L-AT3, which is the group of forms of ATIII that includes both the latent form and the locked form, are similar in conformation to R-AT3, and are also known in the art. Carrell et al., Nature 353, 576-578 (1991); Wardell et al., Biochemistry 36, 13133-13142 (1997). L-AT3, for example, can be produced by limited denaturing and renaturing the AT3 protein under specific temperature conditions, e.g., with guanidium chloride.
Prior to the present invention, AT3 was not known to be associated with angiogenesis. The present invention is, in one embodiment, drawn to a fragment, conformation, derivative or biological equivalent of AT3 that inhibits endothelial cell proliferation, angiogenesis and/or tumor growth in vivo.
In one embodiment, the invention relates to a method of inhibiting tumor growth by delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that inhibits tumor growth, conformational variations of other serpins that inhibit tumor growth, an aggregate form of AT3 that inhibits tumor growth, or a fusion protein of AT3 that inhibits tumor growth. The composition may further comprise a physiologically acceptable vehicle.
The invention further relates to a method of inhibiting endothelial cell proliferation comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that inhibits endothelial cell proliferation, conformational variations of other serpins that inhibit endothelial cell proliferation, an aggregate form of AT3 that inhibits endothelial cell proliferation, or a fusion protein of AT3 that inhibits endothelial cell proliferation. The composition may further comprise a physiologically acceptable vehicle.
The invention also relates to a method of reducing or inhibiting angiogenesis comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that reduces angiogenesis, conformational variations of other serpins that reduce angiogenesis, an aggregate form of AT3 that reduces angiogenesis, or a fusion protein of AT3 that reduces angiogenesis. The composition may further comprise a physiologically acceptable vehicle.
In another embodiment, the invention pertains to a method for identifying an inhibitor of tumor growth or an agent that reduces tumor growth, comprising the steps of inoculating an animal with an appropriate innoculum of tumor cells in each of two suitable inoculation sites; identifying inhibition of growth of a tumor, known as the subordinate tumor, at one inoculation site with concomitant growth of a tumor, known as the dominant tumor, at the other inoculation site; isolating cells from the dominant tumor; and purifying a component which inhibits endothelial cell proliferation and/or angiogenesis from the isolated cells. For example, the component may be purified from conditioned media from the cells. In one embodiment of the invention, the tumor cells are derived from tumors selected from the group consisting of small cell lung cancers and hepatocellular carcinomas. In a particular embodiment, the inoculation sites are the flanks of the animal. In one embodiment the inhibitor of tumor growth is an inhibitor of endothelial cell proliferation. In another embodiment the inhibitor of tumor growth is an inhibitor of angiogenesis. In a further embodiment of the invention, the method further comprises a step of selecting for an animal in which inhibition of the growth of the subordinate tumor by the dominant tumor is substantially complete.
The invention further relates to a method of inhibiting tumor growth comprising delivering or administering an inhibitor of tumor growth identified by the methods described herein to a mammal. In a preferred embodiment the inhibitor of tumor growth is a fragment, conformation, biological equivalent, or derivative of AT3.
It is also within the practice of the invention to use a similar method to identify an agent that reduces or an inhibitor of angiogenesis and/or endothelial cell proliferation. Such a method would also comprise the steps of inoculating an animal with an appropriate inoculum of tumor cells in each of two suitable inoculation sites; identifying inhibition of growth of a tumor, known as the subordinate tumor, at one inoculation site with concomitant growth of a tumor, known as the dominant tumor, at the other inoculation site; isolating cells from the dominant tumor; and purifying a component which inhibits endothelial cell proliferation and/or angiogenesis from the isolated cells. The component may be purified from conditioned media from the cells and in a particular embodiment, the inoculation sites are the flanks of the animal.
The invention further relates to a method of reducing or inhibiting angiogenesis and/or endothelial cell proliferation comprising delivering or administering an inhibitor of angiogenesis and/or endothelial cell proliferation identified by the methods described herein to a mammal. In a preferred embodiment the inhibitor of angiogenesis and/or endothelial cell proliferation is a fragment, conformation, biological equivalent, or derivative of AT3.
The invention also relates to a method of treating a disorder mediated by angiogenesis comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3 in an amount effective to reduce angiogenesis to a mammal. In a preferred embodiment the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that reduces angiogenesis, conformational variations of other serpins that reduce angiogenesis, an aggregate form of AT3 that reduces angiogenesis, or a fusion protein of AT3 that reduces angiogenesis. The composition may further comprise a physiologically acceptable vehicle.
The invention also relates to a method of treating a disorder mediated by endothelial cell proliferation comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3 in an amount effective to inhibit endothelial cell proliferation to a mammal. In a preferred embodiment the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that inhibits endothelial cell proliferation, conformational variations of other serpins that inhibit endothelial cell proliferation, an aggregate form of AT3 that inhibits endothelial cell proliferation, or a fusion protein of AT3 that inhibits endothelial cell proliferation. The composition may further comprise a physiologically acceptable vehicle.
The invention also relates to a method of enhancing angiogenesis comprising delivering or administering a composition comprising an effective amount of an antagonist of a fragment, conformation, biological equivalent, or derivative of AT3 wherein the fragment, conformation, biological equivalent, or derivative of AT3 reduces angiogenesis to a mammal. This method can be used, for example, in wound healing and assisted reproduction techniques as well as in coronary artery surgery and the revascularization/collateralization of peripheral vascular vessels. The composition may further comprise a physiologically acceptable vehicle.
The invention also relates to a method of enhancing endothelial cell proliferation comprising delivering or administering a composition comprising an effective amount of an antagonist of a fragment, conformation, biological equivalent, or derivative of AT3 wherein the fragment, conformation, biological equivalent, or derivative of AT3 inhibits endothelial cell proliferation to a mammal. The composition may further comprise a physiologically acceptable vehicle.
Another embodiment of the invention is a kit for detecting the presence of a fragment, conformation, biological equivalent, or derivative of AT3. The kit may contain primary reagents suitable for detecting the presence of the fragment, conformation, biological equivalent, or derivative of AT3 and optional secondary agents suitable for detecting the binding of the primary reagent to the fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is the L form of bovine AT3, the R form of bovine AT3, the L form of human AT3, or the R form of human AT3.
The invention provides for direct administration of the fragment, conformation, biological equivalent, or derivative of AT3, along with the use of the fragment, conformation, biological equivalent, or derivative of AT3 with or without physiologically acceptable vehicles, including but not limited to viral vectors including adenoviruses, lipids and any other methods that have been employed in the art to effectuate delivery of biologically active molecules.
The invention also provides for the production of a fragment, conformation, biological equivalent, or derivative of AT3 in vivo by the delivery of an enzyme. It is also within the practice of the invention to produce a fragment, conformation, biological equivalent, or derivative of AT3 in vivo by the delivery of a composition that effectuates a conformational change in a serpin.
The invention also relates to pharmaceutical compositions comprising a fragment, conformation, biological equivalent, or derivative of AT3. The composition may be effective for inhibiting tumor growth, angiogenesis, and/or endothelial cell proliferation. In one embodiment, an anti-angiogenic pharmaceutical composition comprises a purified form of AT3 that reduces angiogenesis. In a preferred embodiment the purified form of AT3 is the L form or R form of AT3 or a fragment or sequence which includes the active site or region of the L form or R form of AT3. The composition may further comprise a physiologically acceptable vehicle. The fragment, conformation, biological equivalent, or derivative of AT3 may be an active ingredient in a pharmaceutical composition that includes carriers, fillers, extenders, dispersants, creams, gels, solutions and other excipients that are common in the pharmaceutical formulatory arts.
The invention also provides for a method of delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3 by any methods that have been employed in the art to effectuate delivery of biologically active molecules, including but not limited to, administration of an aerosolized solution, intravenous injection, orally, parenterally, topically, or transmucosally.
The invention also provides for a pharmaceutical composition that comprises compositions to facilitate delivery of therapeutically effective amounts of the fragment, conformation, biological equivalent, or derivative of AT3. The pharmaceutical compositions of the invention may be formulated to contain one or more additional physiologically acceptable substances that stabilize the compositions for storage and/or contribute to the successful delivery of the fragment, conformation, biological equivalent, or derivative of AT3.
Additional features and advantages of the invention will be set forth in the description which follows, and, in part, will be apparent from the description, or may be learned by the practice of the invention. The objectives and other advantages of the invention will be realized and attained by the compounds and methods particularly pointed out in the written description and claims hereof as well as the appended drawings.